Antimicrobial peptides derived from cap18

ABSTRACT

The present invention relates to a group of peptidic compounds which have antimicrobial activity. The compounds also have affinity for toxins and especially for bacterial toxins, such as lipopolysaccharide or lipoteichoic acid. The compounds can be used to manufacture medicaments useful for the treatment of bacterial or fungal infections. The medicaments may be administered systemically or locally.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/658,415having an international filing date of 26 Jul. 2005 which is thenational phase of PCT application PCT/NL2005/000545 having aninternational filing date of 26 Jul. 2005, which claims priority fromEuropean application 04077175.0 filed 28 Jul. 2004. The contents ofthese documents are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to antimicrobial compounds which inhibit or killmicroorganisms including gram-negative bacteria and fungi. In addition,the compounds have affinity for toxins and especially for fungal andbacterial toxins such as lipopolysaccharide (LPS) or lipoteichoic acid(LTA), and which can inhibit or neutralize such toxins. Furthermore, thepresent invention relates to the therapeutic and diagnostic use of thecompounds, and to pharmaceutical compositions comprising the compounds,and methods for their administration.

BACKGROUND OF THE INVENTION

Modern pharmacotherapy has been extremely successful in fightingbacterial infections, which used to be one of the prime causes ofpremature death until the middle of the last century. More recently,however, growing concerns over the wide-spread use of highly effectiveantibiotics have arisen because of the steady increase of bacterialresistance. In fact, over the past 25 years, antibioticresistance—especially multiple resistance to a broad range of antibioticcompounds—has increased in virtually every species of bacteria examined.It is presently believed that the antibacterial agents of the mostadvanced type, which are unaffected by common resistance mechanisms, arethe compounds which use appears to select for multidrug-resistantmutants.

Based on this development, experts recommend to use antibiotics far morerestrictively than in the past, both in agriculture and in humanmedicine. For instance, minor infections—especially those which are noteven typically caused by bacteria, such as the common cold—should not betreated with antibiotics, which should rather be reserved for moreserious conditions. Furthermore, it is necessary to develop novelcompounds for treating bacterial infections with completely differenttypes of pharmacological activity, preferably with some activity whichis independent from bacterial resistance to common antibiotics.

One of the conditions in which the widespread use of antibiotics hasbeen discussed controversially is otitis media, either in its acute formor in its chronic state. It has been shown that the number of patientswith otitis media with effusion (OME), i.e. a type of otitischaracterized by the presence of fluid in the middle ear without thesymptoms of an acute infection, has increased dramatically after theintroduction of antibiotic therapy for early acute otitis media (AOM),suggesting that the antibiotics themselves play a part in OME (Lim etal., Laryngoscope 92, 278-286, 1982). It is believed that antibioticslike penicillin interfere with the development of local immuneresponses, such as with the production of local IgM in the middle ear(Howie et al., Ann. Otol. Rhinol. Laryngol. 85 Suppl. 25,18-19, 1976).Another disadvantage of conventional antibiotic therapy is that thebacteria are killed, but their toxins are still active.

It has been suggested that, for the treatment of these and otherconditions resulting from bacterial or fungal infection, it may beadvantageous to use compounds which do not kill the microorganisms orgerms themselves, but rather neutralize their toxins and allow thenatural host defence mechanisms to control the spread of the infection(Nell, The Role of Endotoxin in the Pathogenesis of Otitis Media WithEffusion, PhD Thesis, Leiden, 1999). At the same time, this strategywould support the rapid restoration of impaired mucosal functions.

A major role among microbial toxins, such as fungal toxins andespecially bacterial toxins, involved in a number of infectiousconditions such as otitis is played by endotoxins, a group oflipopolysaccharides (LPS) found in the cell wall of gram-negativebacteria, consisting of a polysaccharide conjugated with a highly toxiclipid moiety, lipid A. One of the recent therapeutic approaches to treatOME is to administer compounds that neutralize endotoxin, or LPS (Nell,ibid.).

Various compounds capable of neutralizing endotoxin, or LPS, arepresently known. For instance, several anti-endotoxin antibodies havebeen developed, such as HA-1A and E5, a human and a mural monoclonal IgMantibody, respectively. These antibodies have been shown to improvesurvival rates of patients with some severe conditions such as septicshock (Ziegler et al., New Engl. J. Med. 324, 429-436, 1991). However,their activity and specificity is considered unsatisfactory.

Another group of substances active against endotoxin is derived from ahuman endogenous protein termed bacterial permeabihty-increasing protein(BPI), which is stored in the azurophilic granules of neutrophils(Gazzano-Santoro et al., Infection and Immunity 60:11. 4754-4761, 1992).BPI, which is a strongly cationic protein, not only neutralizes freeendotoxins, but also inhibits or kills bacteria cells per se byincreasing the permeability of their outer membranes. BPI is indeed apotent, natural antibiotic, induced by the presence of LPS and someother triggers including tumor necrosis factor (TNF). However, most ofits activity is associated with the immune cells synthesizing it, i.e.polymorphonuclear macrophages.

Several recombinant proteins derived from BPI have also been developed,such as rBPI₂₃ (Kohn et al, 1993) and rBPI₂₁ (Horwitz et al., 1996),which largely represent the N-terminal portions of BPI with molecularweights of 23 and 21 kDa, respectively. The use of BPI and BPI-derivedcompounds in the treatment of OME has, e.g., been described inWO-A-00/71149.

Another family of natural compounds with antimicrobial activity are thecathelicidins, a class of peptides produced by respiratory epithelialcells, alveolar macrophages, and other tissues. In their native forms,these compounds are linear, α-helical, cysteine-free peptides orproteins. Cathelicidins are cationic and comprise a highly conservedsignal sequence and pro-region, cathelin. However, their C-terminaldomain encoding the mature peptide shows substantial heterogeneity. Thepeptides may have 12 to 80 amino acids.

The most prominent human cathelicidin is an 18 kDa cationicantimicrobial protein, CAP18. The 37 C-terminal amino acids of CAP18,i.e. peptide LL-37, represent a domain responsible for the high affinityand neutralizing capacity for LPS (Sawa et al., Antimicr. AgentsChemother. 42:12, 3269-3275, 1998), Several truncated peptides derivedfrom CAP18 or LL-37 have been developed and tested, such as thosedisclosed by Sawa (Sawa et al., ibid.), Gutsmann (Gutsmann et al.,Biophys. J. 80, 2935-2945, 2001), and in U.S. Pat. No. 6,040,291. Ingeneral, relatively small peptides are preferred over proteins such asCAP18 as lead candidates for therapeutical compounds for severalreasons. Firstly, they can more easily be optimized, adapted, andmodified to conserve or augment their desired activity and specificity.Secondly, they are easier to obtain or synthesize, and therefore moreaccessible. Thirdly, they are easier to formulate and deliver, asproteins are often unstable and not bioavailable after non-parenteraladministration.

The co-pending International Patent Application PCT/NL2004/00060, whichis incorporated herein by reference, discloses peptidic compounds whichhave affinity to microbial and fungal toxins such as LPS and LTA. Thecompounds comprise an amino acid sequence X₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆(herein-below, also referred to as core amino acid sequence), wherein X₁represents the N-terminal part of the sequence, X₂ is K or E, X₃ is Q orE, X₄ is D or R, X₅ is N or E, and X₆ represents the C-terminal part;and wherein one or more of the amino acids of the core sequence may bederivatized. The sequence is further characterised in that theN-terminal part is acetylated, and/or that the C-terminal part isamidated, and/or that the amino acid sequence is different fromX₁KEFKRIVQRIKDFLRNLVX₆.

Said patent application further describes methods for the preparation ofsuch compounds. The methods include the chemical and enzymatic ligationof amino acids monomers or oligomers to assemble the compounds. Theyalso include the expression of nucleic acid sequences encoding thecompounds in host cells, using a vector for transacting the host cellswith the nucleic acid sequences. A method for the preparation of acompound according to any one of the previous claims, wherein amino acidmonomers, amino acid oligomers, or mono- or oligomers of amino acidanalogues or mimetics are assembled by chemical or enzymatic ligation,which is performed in a liquid phase and/or at the interface to afunctiohalized solid phase.

These compounds have been found to be useful in the management ofconditions associated with, or resulting from infections. It wassuggested that they may be therapeutically more useful than conventionalantibiotics in the treatment of certain chronic infections, such asotitis media. However, in the case of severe acute infections, effectivecontrol of microbial growth still is considered indispensable, which isachieved by the administration of antibiotics.

OBJECTS OF THE INVENTION

Despite the efforts in the prior art, there is a need for improvementsin the prophylactic respectively therapeutic treatment of infectiousdiseases, including systemic or local bacterial and fungal infections.One of the objects of the invention is to provide new therapies whichare safe, effective, and which lead to an effective control of infectivemicroorganisms. It is another object to provide therapies which do noteasily lead to microbial resistance. Furthermore, it is an object of theinvention to provide therapies which also reduce the undesirable effectsof conventional antimicrobial therapy, such as the toxic effects of themicrobial toxins which are released when microorganisms are killed byantimicrobial compounds in the body.

These and other objects of the present invention will become clear onthe basis of the following description.

SUMMARY OF THE INVENTION

in a first aspect, the present invention provides the use of a peptidiccompound for the manufacture of a medicament for the preventi ortherapeutic treatment of a bacterial or fungal infection of a mammal,wherein the compound comprises an amino acid sequenceX₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆, wherein X₁ represents the N-terminal part,X₂ is K or E, X₃ is Q or E, X₄ is D or R, X₅ is N or E, and X₆represents the C-terminal part. One or more of the amino acids of thecore sequence are optionally derivatized. Furthermore, the N-terminalpart is acetylated, and/or the C-terminal part is amidated, and/or theamino acid sequence is different from the native amino acid sequenceX₁KEFKRIVQRIKDFLRNLVX₆.

Such compounds were surprisingly found to have not only affinity tolipopolysaccharides (LPS) or lipoteichoic acid (LTA), but also directantimicrobial, or microbicidal, activity. That is, these compounds canbe used in the manufacture of medicament that kills bacteria and fungi.The compounds have bactericidal and fungicidal activity. By virtue ofthis activity, the compounds can be therapeutically used as antibiotics,even in diseases and conditions which could not be treated withcompounds capable of neutralising microbial toxins alone. Examples ofsuch diseases are acute bacterial or fungal infections, such as septicshock, acute infections of the eye(s), liver, kidney(s), lungs, bronchi,nasal or frontal sinus, ear(s), vagina, urethra, skin, central nervoussystem, cardiac muscle, spleen, and other organs and tissues.

In a further aspect, the invention relates to medicaments which aresuitable for the prevention and/or treatment of diseases and conditionsresembling bacterial or fungal infections, or being associated withbacterial or fungal infections.

Further aspects of the invention will be set forth in the detaileddescription below and in the appending claims.

DETAILED DESCRIPTION OF THE INVENTION

It was surprisingly found that the peptidic compounds disclosed in theco-pending International Patent Application PCT/NL2004/00060 do not onlyneutralise bacterial and fungal toxins such as lipopolysaccharides (LPS)and lipoteichoic acid (LTA), but also have a significant antimicrobialactivity. These compounds comprise the core amino acid sequenceX₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆, wherein X₁ represents the N-terminal part ofthe sequence, X₂ is K or E, X₃ is Q Or E, X₄ is D or R, X₅ is N or E, X₆represents the C-terminal part; and wherein one or more of the aminoacids of the core sequence may be derivatized. Furthermore, either theN-terminal part of the sequence is acetylated, and/or the C-terminalpart is amidated, and/or the amino acid sequence is different fromX₁KEFKRIVQRIKDFLRNLVX₆. By virtue of the affinity to microbial toxins,the compounds can be therapeutically used to manage conditionsassociated with the presence of such toxins in the body. By virtue oftheir direct antimicrobial activity, however, they can also be used as,or in the place of, antibiotics. The dual activity makes these compoundsextremely useful both when administered alone and in combination withother antibiotics whose undesired side effects, such as the generationof resistant microorganisms or the toxic effects of microbial toxinswhich are released into the body when large numbers of germs are killedwithin the body, may be inhibited or alleviated.

In this description and the appending claims the terms “wherein theN-terminal part is acetylated” have the following meaning. TheN-terminal part is protected by reaction with a carboxyjic acid toobtain an amide linked stabilizing or protecting group. It is, forinstance, possible to react the peptide with fumic acid to obtain aformyl stabilized peptide; with acetic acid to obtain an acetylprotected peptide. Further the peptide can be reacted with propionicacid and other organic acids having up to 6 carbon atoms and even up to10 carbon atoms in the carbohydrate part R. In these organic acids thecarbohydrate group is R having up to 10 carbon atoms, may be straight,or branched, or cyclic and/or may contain ohe or more unsaturations.Moreover, the alkyl chain can be substituted with e.g. hydroxyl,halogen, amino, mercapto and sulphuroxide groups. Hence, at theN-terminal part the following group can be present: —C(O)—R′.Alternatively, instead of reaction with a carboxylic acid, the reactioncan also be carried out with a sulphonic acid to obtain thecorresponding sulfonamide linkage. Hence, at the N-terminal part thegroup —SO₂—R may be present. Alternatively, the said terms alsoencompass alkylation and dialkylation so that at the N-terminal part asecondary or tertiary amine group —N—(R)₁ or N—(R)₂ may be presentwherein each R has the above meaning.

In yet a further embodiment the “acetylation” encompasses reaction ofthe peptide with an isocyanate or an isothiocyanate in which case a ureaor thiourea is created: R—N—C(O)— or R—N—C(S)— respectively, R being asdefined hereinabove.

Finally, the N-terminus can be protected by an acid stable blockinggroup, which group is conventionally introduced during peptidesynthesis, but will now not be removed. Well-known blocking groups arethe F_(moc) and Z-group.

As to the meaning of the terms “wherein the C-terminal part is amidated”the following is noted. The term “amidation” means that the —OHnaturally present as the C-terminus is replaced by the group —X, whereinX is (i) a —NY₂ group, Y, independently being H, or R, wherein R is asdefined hereinabove or the two Y-groups together may be a cyclic moietytogether with the N-group to which they are attached, preferably atleast one R group is present; (ii) an —OR group wherein R is as definedhereinabove, or (iii) a —R group. The peptide amides are preferredbecause these have the highest stability.

The peptidic compounds of the present invention have been found to havean optimized stability compared to the native amino acid sequenceexcluded from claim 1.

Peptidic compounds are peptides, such as oligo- of polypeptides,proteins, or substances derived from peptides. Beyond peptidesthemselves, peptidic compounds also encompass analogues of peptides,peptide derivatives, modified peptides, and peptide conjugates. Peptidiccompounds have in common that they comprise amino acid sequences. Moreprecisely, peptides are defined as amides that are derived from two ormore amino acids by combination of the amino group of one acid with thecarboxyl group of another (Merriam Webster Medical Dictionary 2001). Apeptidic compound, in contrast, may also refer to a peptide structurewithin a molecule. Typically, peptides are composed of naturallyoccurring (L-)α-amino acids, in particular alanine (Ala or A), arginine(Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Glyor G), histidine (His or H), isoleucine (IIe or I), leucine (Leu or L),lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or P),proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan(Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Analogues or functional equivalents of peptides are peptidic molecules,comprising the same activity and especially the same affinity tomicrobial and especially to bacterial toxins in kind, but notnecessarily in amount, and may, for instance, be modified peptides,peptoids, peptide analogues or peptidomimetics.

Modified peptides are molecules derived from peptides by theintroduction of substituents or functional groups which are, generally,not present in naturally occurring amino acids. The term also includescompounds which are obtained by the reaction of peptides with moleculesfrom other chemical categories, whether these molecules are naturallyoccurring or not. For instance, phosphorylated, sulfonated andbiotinylated peptides, glycoproteins, and lipoproteihs are frequentlyfound in nature, while peptides modified with polyethylene glycol areexamples of chemically modified peptides that have been designed toalter some, but not all of the peptides properties.

Peptoids, like peptides, aire also peptidic compounds. They are alsotypically amides of two or more amino acids. However, they arefrequently not directly derived from naturally occurring amino acids,but rather of various types of chemically synthesized L- and/or D-aminoacids.

Peptidomimetics, in their broadest scope, are compounds which are intheir functional structure more or less similar to a peptide, but whichmay also contain non-peptidic bonds in the backbone, or D-amino acids.In general, peptidomimetics serve as substitutes for native peptides inthe interaction with receptors and enzymes (PharmaceuticalBiotechnology, Ed. D. J. A. Crommelin and R. D. Sindelar, HarwoodAcademic Publishers, 1997, p. 138). Pseudopeptides, a class ofpeptidomimetics, are compounds containing amide bond isosteres insteadof amide bonds (ibid., pp. 137-140).

The compounds which are used to carry out the invention also includesalts of peptides or functional equivalents, such as pharmaceuticallyacceptable acid- or base addition salts, and adducts. They also includemultimers of peptides or functional equivalents.

Furthermore, the compounds have affinity to at least one toxin, andespecially a bacterial toxin. In a large number of infectious diseases,bacterial toxins, such as the class of lipopolysaccharides (LPS) in thecase of gram-negative bacteria, and lipoteichoiec acid in the case ofgram-positive bacteria, are involved in the manifestation of thedisease. These toxins can induce substantial inflammatory reactions. Forinstance, in upper airway infections, the inflammation may lead tomucosal damage of the epithelial of the middle ear or the sinuses,resulting in the impairment of the mucociliary clearance system (MCS),which is one of the major defence systems of the upper airways. Affinityto the fungal or bacterial toxins is a prerequisite of anyneutralization capability, and preferably, the compounds of theinvention not only bind to LPS and other toxins, but also have theability to neutralize or inhibit these toxins or otherwise reduce theeffects of said toxins.

The desired type of activity against bacteria and fungi, and bacterialand fungal toxins, is observed when peptidic compounds fulfil thestructural requirements as defined in claim 1, according to which thecompounds comprise an amino acid sequence X₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆,wherein X₁ represents the N-terminal part of the sequence, X₂ is K or E,X₃ is Q or E, X₄ is D or R, X₅ is N or E, and X₆ represents theC-terminal part. This basic motif is derived from the naturalantimicrobial protein CAP18, or the peptide LL-37which is itself derivedfrom CAP18.

As used herein, the N-terminal part is a group, atom, or sequencerepresenting the N-terminal moiety or domain of the compound, i.e. thestructure that is attached to the terminal α-amino group of the coresequence which is not involved in an amide bond within the sequence. TheN-terminal part may simply be a hydrogen atom in the case of a freeα-amino group; or it may consist of a chemical group attached to theterminal α-amino nitrogen atom, such as an acyl group. It may alsorepresent a larger group, such as a sequence of two or more amino acids,or a chemical structure which is not composed of or not solely composedof amino acids. The G-terminal part is defined in an analogue fashion.

Preferably, the compounds comprise a total of more than the 18 aminoacids defining the core motif. In one embodiment, the N-terminal partcomprises a sequence of two or more amino acids. Among the amino acidswhich are suitable members pf this sequence are I and G, and a preferredN-terminal part is IG.

In another embodiment, the C-terminal part also comprises an amino acidsequence. The sequence may comprise 1, 2, 3, 4, or more than 4 aminoacids. In one embodiment, the C-terminal part comprises 4 amino acids.The C-terminal end of said C-terminal part of 4 amino acids may be an E,as in the equivalent position within peptide LL-37. However, thisC-terminal end may also be defined by an R. The amino acid which ispositioned next to the C-terminal amino acid may be T as in LL-37, or itmay be L. P and R are two other preferred members of the 4 amino acidsequence of the C-terminal part, in either of the two remainingpositions. Most preferably, the C-terminal part is selected from thesequences PRTE and RPLR.

In a further embodiment, the N-terminal part and the C-terminal part areselected from the preferences described above to yield a peptidicstructure with a total number of 24 amino acids. Among the presentlymost preferred compounds are the peptides IGKEFKRIVQRIKDFLRNLVPRTE andIGKEFKRIVERIKRFLRELVRPLR, either as peptides themselves, or as modifiedor derivatized peptides.

Among the preferred modifications are amidated and/or acetylatedpeptides. One of the positions in which amidation seems particularlyadvantageous is the C-terminus of the peptide. Acetylation, on the otherhand, is preferably performed at the N-terminal amino acid. In one ofthe presently preferred embodiments, the peptidesIGKEFKRIVQRIKDFLRNLVPRTE and IGKEFKRIVERIKRFLRELVRPLR are bothN-terminally acetylated and C-terminally amidated. Preliminary testingsuggested that these modifications possess an increased stability in thepresence of exo-peptidases.

The compounds can generally be prepared by methods that are known forthe preparation of peptides and similar substances. Smaller compoundscontaining only a few amino acids or similar units, and preferably notmore than 30-50 units, can be prepared by chemical or enzymatic ligationtechniques, either using the classical approach in which the reactionstake place in solution or suspension, or by employing the more modernsolid phase approach, in which the peptide is assembled while beinganchored to a solid surface, such as a polymeric bead. Larger compoundsare typically synthesized by automatic solid phase peptide synthesizers.

Alternatively, the compounds can be prepared by known geneticengineering techniques. This approach is especially valid if thecompound is indeed a peptide or a slightly modified peptide. Forinstance, a DNA sequence which encodes the compound can be associated orcombined with an expression vector capable pf transfecting cells. Inanother step of the method, host cells or target cells are transfectedwith said DNA by contacting the cells with the vector and thevector-associated DNA under conditions which allow transfection. In afurther step, the host or target cells are cultured under conditionswhich allow the expression of the compound. Subsequently, the compoundcan be isolated. If the compound itself cannot be encoded or expressedbut is very similar to a peptide that can be encoded or expressed, themethod can be applied to prepare the peptide to which the compound issimilar, followed by one or more steps wherein the peptide is modifiedby chemical or enzymatic techniques to prepare the compound.

Various types of vectors are used for this purpose, such as viralvectors, lipoplexes, polyplexes, microspheres, nanospheres, dendrimers,naked DNA, peptide delivery systems, lipids, especially cationic lipids,or liposomes made thereof, polymeric vectors, especially those made ofpolycationic polymers. Among the preferred viral vectors areretroviruses, adenoviruses, adeno-associated viruses, herpes simplexviruses, and virosomes. Preferred non-viral vectors include chitosan,SPLP, polymeric systems based on PLGA, polyethylene imines, polylysines,polyphosphoamidates, poly(meth)acrylates, polyphosphazenes, DOPE, DOTAP,and DOTMA.

Some more comprehensive summaries of methods which can be applied in thepreparation of the compounds of the invention are described in: W. F,Anderson, Nature 392 Supp,, 30 Apr. 1998, p. 25-30; PharmaceuticalBiotechnology, Ed. D. J. A. Crommelin and R. D. Sindelar, HarwoodAcademic Publishers, 1997, p. 53-70, 167-180, 123-152, 8-20; ProteinSynthesis: Methods and Protocols, Ed. R. Martin, Humana Press, 1998, p.1-442; Solid-Phase Peptide Synthesis, Ed, G. B. Fields, Academic Press,1997, p. 1-780; Amino Acid and Peptide Synthesis, Oxford UniversityPress, 1997, p. 1-89.

Salts of peptides pr functional equivalents are prepared by knownmethods, which typically involve the mixing of the peptide or peptoidwith either a pharmaceutically acceptable acid to form an acid additionsalt, or with a pharmaceutically acceptable base to form a base additionsalt. Whether an acid or a base is pharmaceutically acceptable can beeasily decided by a person skilled in the art after taking the specificintended use of the compound into consideration. For instance, not allacids and bases that are acceptable for in vitro diagnostic compositionscan be used for therapeutic compositions. Depending on the intended use,pharmaceutically acceptable acids include organic and inorganic acidssuch as formic acid, acetic acid, propionic acid, lactic acid, glycolicacid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonicacid, cinnamic acid, sulphuric acid, hydrochloric acid, hydrobromicacid, nitric acid, perchloric acid, phosphoric acid, and thiocyanicacid, which form ammonium salts with free amino groups of peptides andfunctional equivalents. Pharmaceutically acceptable bases, which formcarboxylate salts with free carboxylic groups of peptides and functionalequivalents, include ethylamine, methylamine, dimethylamine,triethylamine, isopropylamine, diisopropylamine, and other mono-, di-and trialkylamines, as well as arylamines. Moreover, alsopharmaceutically acceptable solvates, complexes or adducts, such ashydrates or ethurates are encompassed.

Some of the preferred modifications of the peptides may be easilyintroduced during or at the end of the synthesis. For instance, when thepeptide is synthesized using a solid-phase technique, N-terminalacetylation can be performed at the end by reacting the amino acidsequence, which is still bound to the resin, with acetic acid instead ofwith another amino acid.

C-terminal amidation, on the other hand, can be performed by using aspecial kind of resin in solid-phase peptide synthesis, such as thecommercially available Tentagel S AM (ex Rapp, Tübingen, Germany). Theseresins comprise a chemical “handle” from which amidated peptides arereleased during the cleavage. These and further methods of modifyingpeptides are known to any person skilled in the art.

As previously mentioned, the compounds have an affinity to microbialtoxins and especially to bacterial toxins, such as lipopolysaccharide(LPS) and lipoteichoic acid (LTA). Therefore, the compounds can be usedadvantageously for preventive, therapeutic and diagnostic purposes inconditions and diseases in which the presence of these toxins isinvolved. The binding ability will typically lead to neutralization ofthe toxins, by virtue of which the compounds may be consideredantagonists or partial antagonists. Furthermore, they may be used astargeting agents or ligands for other compounds which are capable ofneutralizing the toxins, and which may be specifically targeted to thesetoxins through covalent or non-covalent ligation with the compounds, orthrough being covalently or non-covalently bonded to the surface of adrug carrier such as a liposome, a nano- or microparticle, a nano- ormicrocapsule, a lipid complex, or a micelle.

In diagnosis, the compounds may be used for the detection of, or thequantification of the amount of, bacterial toxins present inphysiological fluids, such as the blood, plasma, serum, the mucus lininga mucosal epithelium, such as of the respiratory tract, or in fluidswhose presence results from a pathological condition, such as the fluidfound in the middle ear in otitis media with effusion (OME). For thisuse, the compounds may be incorporated into diagnostic kits to be usedin vitro, or into diagnostic compositions which may be administered to apatient. For this use, an option is to conjugate a compound of theinvention with a chelator, which is subsequently complexed with anisotopic label that is detectable by an appropriate monitoring system.

In a preferred use, the compounds are administered as active drugsubstances to prevent or treat diseases and conditions related to fungaland bacterial infections and the presence of fungal and bacterial toxinsin the body. As mentioned before, there are certain disadvantages andlimitations of conventional antibiotics in the therapy of acute orchronic infections, such as the induction of tolerance and the selectionof tolerant bacterial variants, the depression of the patient's naturaldefence systems, the impairment of the bacterial flora naturallypopulating the mucosae, the release pf large amounts pf bacterial toxinsas the germs are killed etc. Furthermore, there may be conditions anddiseases in which the presence of toxins and especially bacterialtoxins, and not the presence of the micro-organisms per se, is the majorcause, such as in OME, wherein the local retention of toxins in themiddle ear may significantly contribute to the manifestation of thedisease even in the absence of symptoms of an acute infection.

However, since the compounds also have pronounced antimicrobialactivity, they can be used even in those conditions in which it isessential to actually reduce the number of microorganisms infecting thebody, such as severe acute infections. In this respect, they can replaceor complement other antibiotics, even in diseases and conditions whichcould not be treated with compounds capable of neutralising microbialtoxins alone. Examples of such diseases are acute bacterial or fungalinfections, such as septic shock, acute infections of the eye(s), liver,kidney(s), lungs, bronchi, nasal or frontal sinus, ear(s), vagina,urethra, skin, central nervous system, cardiac muscle, spleen, or othertissues and organs. An example of a particularly severe conditionassociated with an infection is septic shock.

In all these cases, it may be advisable to treat the disease not withantibiotic drugs, but with substances which are capable of neutralizingthe bacterial toxins. For this aim, the compounds of the invention areparticularly advantageous because they show a high binding andneutralization activity against the most relevant microbial toxins, suchas lipopolysaccharide (LPS) in the case of gram-negative bacteria, andlipoteichoic acid (LTA) in the case of gram-positive bacteria. Ininfections of the upper airways, for the treatment of which thecompounds of the invention are particularly preferred, these bacterialproducts can induce an inflammation reaction in the middle ear or in thesinuses, and can induce mucosal damage of the upper airway epithelia.Neutralizing the toxins involved may allow the mucosal damage includingthe impairment of the mucociliary clearance system (MCS) to beprevented, controlled, or reduced, and will thus strengthen the naturaldefence systems. In those cases including OME, in which bacterial toxinsmay represent this major problem even in the absence of significantnumbers of living bacterial cells, a therapy relying on theadministration of a compound of the invention, for instance directly tothe middle ear, may represent the primary therapeutic approach. But alsoin other airway infections, such as acute or chronic sinusitis, or acuteor chronic otitis, the compounds may be highly useful for therestoration of the normal mucosal functions and their natural defencesystems.

More generally speaking, the compounds of the invention are usefulagents in the prevention and therapy of conditions consisting in, andarising from, infective bacteria iacluding Streptococcus pneumoniae,Haemophilus influenzae, Moraxella catarrhalis, group A β-hemolyticstreptococci. Staphylococcus aureus, gram-negative enteric bacilli,Streptococcus pyrogenes, Escherichia coli, gram-negative bacilli,Pseudomonas sp.

The infections which are prevented or managed with the compounds of theinvention may be caused by microorganisms which are in essenceplanktonic, that is, their habitus is that of individual microbialorganisms. If planktonic microorganisms, for example, have infected aphysiological fluid of a mammal such as blood, they may be transportedwith the bloodstream throughout the body of the mammal.

Systemic or generalised infections which may at least in part be causedby planktonic microorganisms include acute or chronic fungal infectionssuch as actinomycosis, blastomycosis, nocardiosis, cryptococcosis,sporotrichosis, sporotrichosis, coccidiomycosis, and aspergillosis, aswell as acute or chronic bacterial infections such as anthrax, tetanus,gangrene, botulism, listeriosis, typhus, Legionnaire's disease, cholera,yellow fever and the like. Acute serious systemic infections may beassociated with septic shock, which is a life-threatening severe form ofsepsis that usually results from the presence of large numbers ofgram-negative bacteria and their toxins in the bloodstream, and which isfurther characterised by decreased blood flow to organs and tissues,hypotension, organ dysfunction, impaired mental state, and oftenmultiple organ failure, and which often affects immunocompromisedindividuals.

In more recent years, however, it has been found that many infectiousdiseases and conditions are also, at least in part, caused bymicroorganisms which form more sessile communities, usually referred toas biofilms. This is true both for many systemic and—in particular—for alarge number of more localised infections. As used herein, a biofilm isa microbially derived sessile community characterised by cells that areattached to a substratum or interface or to each other, are embedded ina matrix of extracellular polymeric substances that they have produced,and exhibit an altered phenotype with respect to growth rate and genetranscription. Usually, the extracellular matrix comprises a highlyhydrated, predominantly anionic matrix polymer. Biofilms can adhere tosurfaces and interfaces; in fact, adhesion may trigger the expression ofgenes controlling the production of the extracellular matrix and theconversion of the previously planktonic microorganisms into theirsessile phenotypes.

It is believed that sessile microorganisms and their biofilms play amajor role in a number of infectious diseases, and for some of them thishas been supported by a large body of evidence. Among these diseases aree.g. periodontitis, native valve endocarditis, cystic fibrosis, chronicbacterial prostatitis, bronchitis, pneumonia, sinusitis, dental caries,chronic tonsilitis, endocarditis, necrotising fascitis, musculosceletalinfections, osteomyelitis, biliary tract infections, infectious kidneystones, and otitis media. Most likely, many other—in particularlocal—infections involving particular regions or organs of the body alsoinvolve sessile microorganisms, such as infections of the liver, thespleen, the periodontium, an eye, a kidney, the skin, the vagina, theurethra, or the heart.

In the investigation of the activity of the peptidic compounds asdefined in claim 1, it has now been found that these compounds are alsoactive against sessile, bipfilm-forming microorganisms. Thus theyrepresent useful compounds for the prevention or therapy of diseasesrelated to such microorganisms, or involving the sessile state ofmicroorganisms. For example, it has been found that certainmicroorganisms capable of forming biofilms on certain surfaces such asPVC, e.g. Pseudomonas putida, could be inhibited and prevented fromforming biofilms in a standard biofilm assay.

The inhibition effect may be achieved when a compound of the inventionis present at a concentration of at least about 0.001 μM, and morepreferably of at least about 0.01 μM, and still more preferably at leastabout 0.1 μM. In a further preferred embodiment, the concentration ofthe compound is about 0.1 to about 100 μM. However, depending on thetype and amount of fluid which contacts the biofilms, and the specificmicroorganism involved, these concentrations may be adapted.

Furthermore, it has been found that the compounds of the invention notonly prevent the formation of biofilms by microorganisms otherwisecapable of microfilm formation, but are also active against microfilmsthat have formed already. Depending on their concentration, they arecapable of disrupting or degrading biofilms, as detectable e.g. instandard biofilm assays. Again, the concentration of the compound shouldbe selected by taking into account the type and amount of fluid whichcontacts the biofilms and the specific microorganism involved. Forexample, it has been found that concentrations as low as about 0.001 μMmay already have a disruptive effect on pre-formed biofilms. Thus, it ispreferred that the concentration of the compound is selected to be atleast about 0.001 μM. Other preferred concentrations are at least about0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM, respectively.

A particular risk from biofilm infectious arises from the insertion orimplantation of medical devices in the body of a human, or other mammal,whether for diagnostic or therapeutic purposes. For example, theinvolvement of biofilms has been demonstrated for infections fromcontact lenses, heart valves, venous catheters, urinary catheters,intrauterine devices, sutures, vascular grafts and shunts, peritonealdialysis devices, penile prostheses, and orthopaedic prostheses. Thus,it is another preferred embodiment to use the compounds of the inventionto prevent or manage an infection resulting from such devices.

A particular advantage of the compounds of the invention over the nativeproteins and peptides from which they are derived, such as CAP18 andLL-37, is their low degree of undesirable inflammatory activity. Thisactivity is related to the various cellular processes, includingproliferation, differentiation and expression of genes encodingpro-inflammatory mediators like cytokines. Cytokines are directmediators of inflammation and influence the progress and direction ofmany immunological reactions. Perturbation of the balance in cytokineproduction is widely recognized as a critical factor in several diseasestates. In a condition such as otitis media with effusion or sinusitis,this balance is already disturbed. T cell proliferation is also notfavourable in this situation, because this will further stimulate theimmune response that is already out of control.

Thus, the compounds can be advantageously used in pharmaceuticalcompositions. According to the invention, such pharmaceuticalcompositions are provided as well as the compounds themselves. As usedherein, the term “pharmaceutical composition” refers to therapeutic anddiagnostic compositions, as well as to medicaments and diagnosticscontaining such compositions. Therapeutic compositions and medicamentsare used for the prevention or treatment of diseases and otherconditions of mammals whose improvement is desirable. Diagnostics anddiagnostic compositions are used for the diagnosis of such diseases invivo and in vitro.

Typically, such a medicament, or composition, comprises at least onecompound of the invention as active ingredient and at least onepharmaceutically acceptable carrier or excipient.

In one of the embodiments, the medicament comprises another activeingredient, which may be selected from the same group of compounds asspecified in claim 1. Alternatively, the other compound belongs to adifferent group. For example, it may be a compound also known to haveantibiotic or antifungal activity, but with a different mechanism ofaction. In a further embodiment, the other compound is not incorporatedinto the same medicament, but co-administered as a separate formulation.

Furthermore, the composition is processed and shaped in such a way thatit can be administered to a human being, or to an animal. As usedherein, a carrier or excipient is any pharmaceutically acceptablesubstance or mixture of substances having no substantial pharmacologicalactivity, which can be used as a vehicle or as an auxiliary substance toformulate a compound into dosage form which is stable and suitable toadniinister. Examples of pharmaceutically acceptable excipients areknown to the skilled man and can be found in the monographs of the majorpharmacopoeias.

In one embodiment, the composition is formulated and processed forparenteral injection, instillation or irrigation, preferably forintravascular injection, such as intravenous or intra-arterial, but alsofor intramuscular, subcutaneous, intralesional, intraperitoneal,locoregional or other routes of parenteral administration. In anotherpreferred embodiment, the composition is administered directly to theaffected mucosa of the upper airway, such as the middle ear. The sameprinciples that govern the formulation of other drugs for theseadministration routes will also teach those skilled in the arts how toprepare such compositions. For instance, one of the requirements ofparenteral dosage forms is their sterility. Other requirements aredescribed in all major pharmacopoeias, such as in USP 24, in themonograph “General Requirements for Tests and Assays. 1. Injections”, p.1775-1777. To increase the stability of a parenteral formulation, it maybe necessary to provide a dried dosage form which must be reconstitutedbefore it can be administered. An example of such a dosage form is afreeze-dried or lyophilized formulation. Suitably, the compositions ofthe invention may also contain a mucolytic solvent.

It may be desirable to administer a compound of the invention as aparenteral controlled release dosage form to avoid frequent injectionsand to improve the effectiveness and convenience of the therapy. Variousmethods of preparing such depot formulations are known. Prolongedrelease may be provided by solid implants, nanoparticles, nanocapsules,microparticles, microcapsules, emulsions, suspensions, oily solutions,liposomes, or similar structures.

In the case of compositions which are to be administered locally to anaffected mucosa, it may be useful to provide a formulation havingproperties which provide for an extended time of local retention at thesite of administration to increase the effectiveness of the medication.To achieve this goal, mucoadhesive excipients may be incorporated intothe formulation. Such functional excipients are known to the personskilled in the art; they include polymers such as polyacrylic acids andderivatives thereof, polymethacrylic acids and their derivatives,cellulose ethers including hydroxypropyl methylcellulose,carboxymethylcellulose, starches, chitosan etc. Suitably oralternatively, the compositions of the invention may also contain amucolytic solvent. Particularly, mucolytic solvents are used to affectthe permeability of the peptidic compound of the invention into themucus, e.g. in the respiratory tract. Suitable solvents may compriseknown mucoregulatory or mucolytic agents such as N-acetylcysteine,S-carboxymetbyl cysteine, bromhexine, ambroxyl, DNAse, erdosteine,saline solution and nesosteine. Preferably, bromhexine is used.

Further excipients that are particularly useful for the preparation ofparenteral formulations in their broadest definition are solvents,cosolvents and liquid or semisolid carriers, such as sterile water,ethanol, glycerol, propylene glycol, polyethylene glycol, butanediol,fatty oils, short- and medium chain triglycerides, lecithin,polyoxyethylene castor oil derivatives; substances to adjust theosmolality and pH, such as sugars, especially glucose, sugar alcohols,especially mannitol, sodium chloride, sodium carbonate, citric acid,acetate, phosphate, phosphoric acid, hydrochloric acid, sodium hydroxideetc.; stabilizers, antioxidants, and preservatives, such as ascorbicacid, sodium sulphite or -hydrogen sulphite, EDTA, benzyl alcohol etc.;other excipients and lyophilization aids, such as albumin, dextran etc.

Similarly, it may he advantageous to administer a compound of theinvention in a transmucosal dosage form, This route of administration isnon-invasive and patient-friendly; at the same time it generally leadsto an improved bioavailability of the compound of the invention ascompared to oral administration, especially if the compound is notstable in the fluids of the digestive system, or if it is too large tobe absorbed from the gut effectively. Transmucosal administration ispossible, for instance, via nasal, buccal, sublingual, gingival, orvaginal dosage forms. These dosage forms can be prepared by knowntechniques; they can be formulated to represent nasal drops or sprays,inserts, films, patches, gels, ointments, or tablets. Preferably, theexcipients used for a transmucosal dosage form also include one or moresubstances providing for mucoadhesion, thus prolonging the contact timeof the dosage form with the site of absorption and thereby potentiallyincreasing the extent of absorption.

Alternatively, the pharmaceutical compositions may be designed for oraladministration and processed accordingly. Appropriate oral dosage formsinclude tablets, hard capsules, soft capsules, powders, granules, orallydisintegrating dosage forms, syrups, drops, suspensions, effervescenttablets, chewable tablets, oral films, lyophilized dosage forms,sustained release dosage forms, controlled release dosage forms. In oneof the preferred embodiments, the oral dosage form is an entericallycoated solid dosage form to provide protection of the compound from theacidic and proteolytic environment of the stomach.

In a further embodiment, the compounds are administered via thepulmonary route, using a metered dose inhaler, a nebulizer, an aerosolspray, or a dry powder inhaler. Appropriate formulations can he preparedby known methods and techniques. Transdermal, rectal, or ocularadministration may also be feasible in some cases.

It can be advantageous to use advanced drug delivery or targetingmethods to deliver a compound of the invention more effectively. Forinstance, if a non-parenteral route of administration is chosen, anappropriate dosage form may contain a bioavailability enhancing agent,which may be any substance or mixture of substances which increases theavailability of the compound. This may be achieved, for instance, by theprotection of the compound from degradation, such as by an enzymeinhibitor or an antioxidant. More preferably, the enhancing agentincreases the bioavailability of the compound by increasing thepermeability of the absorption barrier, which is typically a mucosa.Permeation enhancers can act via various mechanisms; some increase thefluidity of mucosal membranes, while others open or widen the gapjunctions between mucosal cells. Still others reduce the viscosity ofthe mucus covering the mucosal cell layer. Among the preferredbioavailability enhancers are amphiphilic substances such as cholic acidderivatives, phospholipids, ethanol, fatty acids, oleic acid, fatty acidderivatives, EDTA, carbomers, polycarbophil, and chitosan.

Making use of the antimicrobial and antifungal activity of thecompounds, it is an option to use them for the manufacture ofmedicaments which either contain no preservatives or only at a reducedcontent. By “reduced content” is meant that the preservative content islower than the preservative content needed to effectively preserve thecorresponding placebo composition, which is a composition which containsthe same components except for the active ingredient.

Whether a composition is effectively preserved can be determined withappropriate tests, such as the test for preservative efficacy (e.g. USP<51>), wherein five challenge organisms are tested at defined timeintervals, depending on the product category. Conducted in appropriateseries, such testing can also be performed in order to determine theminimally effective concentration of a specific preservative for a givencomposition, such as a drug-free composition corresponding to acomposition as described above.

For example, it may be found that in order to effectively preserve aparticular placebo composition with sorbic acid, the preservative mustbe present at a concentration of at least about 0.1% (w/v). In thiscase, the reference composition which comprises the compound specifiedin claim 1 could contain sorbic acid at a substantially lowerconcentration, such as about 0.05% (w/v) or less. In another embodiment,the concentration of the preservative is selected to be not more thanabout a fifth, and more preferably not more than about a tenth, of theconcentration needed to effectively preserve a corresponding placebocomposition.

The following examples are intended to further illustrate the invention,not to limit its scope to the embodiments presented herein.

EXAMPLE 1 Preparation of Compounds

The following peptidic compounds, each of them comprising 24amino acids,the compounds herein coded as P60, P60.4, P60.Ac, and P60.4Ac wereprepared by solid phase strategies on an automated multiple peptidesynthesizer (SyroII, MultiSyntech, Witten, Germany), For P60 and P60.4,Tentagel S AC (Rapp, Tübingen, Germany), a graft polymer of polyethyleneglycol and polystyrene was used as a resin (loading 0.2 meq, particlesize 90 μm), For P60.Ac and P60.4Ac, Tentagel S AM was used, whichyields a C-terminally amidated peptide. Repetitive couplings wereperformed by adding a six fold molar excess (based on the resin loading)of a 0.60 M solution of the appropriate Fmoc amino acid in NMP, a sixfold molar excess of 0.67 M PyBOP in NMP and a twelve fold molar excessof NMM in NMP 2/1 (v/v) to the reaction vessel. Side chain protectionwas as follows: tBu for D, E, S, T; Boc for K; Trt for N, Q and Pmc forR. Fmoc-deprotection was performed by adding 3times piperidine/NMP ¼(v/v) to each reaction vessel. Coupling and deprotection times were 45min and 3 times 3 min, respectively. Washings after couplings andFmoc-deprotections were performed 6 times with NMP. For P60.Ac andP60.4Ac, the N-terminal acetylation was performed with acetic acid whilethe peptide was still bound to the resin. After synthesis the peptidylresins were washed extensively with NMP, dichloromethane,dichloromethane/ether 1/1 (v/v) and ether respectively, and air dried.Peptidyl resins were then cleaved and side chain deprotected inTFA/water 95/5 (v/v) for 2.5 h.(1.5 ml per 10 μmol of peptide), theresin was removed by filtration and the peptide was precipitated fromthe TFA solution with ether/pentane 1/1 (v/v) (10 ml per 10 μmol ofpeptide). The solution was cooled for 1 h at −20° C. and theprecipitated peptide was isolated by centrifugation (−20° C., 2,500 g,10 min). After triturating and vortexing of the pellet with 10 mlether/pentane 1/1 (v/v) and isolation by the same procedure, thepeptides were air dried at room temperature for 1 h. Peptides weredissolved in 2 ml water or 2 ml 10 vol % acetic acid, the solution wasfrozen in liquid nitrogen for about 5 min and subsequently lyophilizedwhile being centrifuged (1,300 rpm, 8-16 h). The analysis of thepeptides was performed with RP-HPLC and Maldi-Tof mass spectrometry.

The amino acid sequences of the compounds are:

P60 IGKEFERIVQRIKDFLRNLVPRTE P60.Ac* IGKEFKRIVQRIKDFLRNLVPRTE P60.4IGKEFKRIVERIKRFLRELVRPLR P60.4Ac* IGKEFKRIVERIKRFLRELVRPLRThe suffix Ac means that the peptide is N-terminally acetylated and C-terminally amidated.

EXAMPLE 2 Neutralization of Toxins

The compounds prepared according to example 1 were tested for theircapability to neutralize the bacterial toxin LPS with a limulusamoebocyte lysate (LAL) assay and with a whole blood (WB) assay. LTAneutralization was also measured with a whole blood assay. Peptide LL-37was used as a positive control. The peptide concentration whereby 50%LPS is neutralized was used as a measure of the peptide's activity.These concentration values were as in table 1. The differences betweenthe compounds within each assay were not statistically significant. Insummary, the tested compounds of the invention showed approximately thesame degree of anti-toxin activity as the native antimicrobial peptideLL-37.

TABLE 1 Compound concentrations for 50% LPS- and LTA- neutralization (inμg/ml ± SD) Compound LPS (LAL) LPS (WB) LTA (WB) P60 1.5 ± 0.5 1.4 ± 0.12.1 ± 0.7 P60.Ac 1.8 ± 0.8 2.4 ± 0.5 2.1 ± 0.1 P60.4 1.7 ± 0.6 2.1 ± 0.62.0 ± 1.3 P60.4Ac 1.8 ± 0.1 nd Nd LL-37 (control) 1.3 ± 0.2 1.2 ± 0.31.6 ± 0.5

EXAMPLE 3 Immunologic Cell Activation by Compounds

The compounds prepared according to example 1 were tested for theirtherapeutically undesirable immunogenic activity by using Elispot,T-cell proliferation, ERK-activation, and neutrophil chemotaxis assays.The Elispot assay is applicable to determine effects of drugs, chemicalsor other compounds on cytokine secretion in vitro, thereby providingdata on their putative modulatory effects on immune function in vivo.The results of the assay are given as fraction of positive responses toIFN-gamma. ERK-(extracellular signal-related kinases)-½ is part of theMAP-kinase signaling pathway, that has been shown to be involved invarious cellular processes, including proliferation, differentiation andexpression of genes encoding pro-inflammatory mediators like cytokines.Cytokines are direct mediators of inflammation and influence theprogress and direction of many immunological reactions. Perturbation ofthe balance in cytokine production is widely recognized as a criticalfactor in several disease states. This balance is already disturbed inthe case of conditions such as otitis media with effusion and sinusitis.T. cell proliferation is also not favorable in this situation, becausethis will also stimulate the immune response that is already out ofcontrol. It is therefore desirable that the compounds of the inventiondo not stimulate cytokine production, T cell proliferation,ERK-activation or chemotaxis of neutrophils.

For T cell proliferation, 150,000 peripheral blood mononuclear cells(PBMC) were cultured in the absence or presence of 10 μg/ml of thecompounds for 5 days in 96 well round bottom plates (Costar Inc.Cambridge, Mass.) in a final volume of 150 μl IMDM complete. As apositive control, PBMC were cultured in the presence of 25 U/mlrecombinant IL-2. During the final 20 hours of culture, PBMC were pulsedwith [3H]thymidine (0.5 microCi/well), after which 3H-incorporation wasmeasured by liquid scintillation counting. For detection of the T cellcytokines IFN and IL-10 by Elispot analysis, 1.5×10⁶ PBMC were culturedin 0.5 ml IMDM complete in the absence or presence of variousconcentrations of synthetic peptide. As a positive control PBMC werestimulated by 10 μg/ml poke weed mitogen (PWM). After 48 hours ofculture, PBMC were harvested by gently rinsing the wells with warm IMDMto collect non-adherent cells, which were washed in a large volume ofIMDM. PBMC were subsequently plated on antibody-precoated ELISA platesand cultured for 5 hours in IMDM supplemented with 2% pooled human ABserum at 37° C. 5% CO₂, after which the plates were developed accordingto the manufacturer's protocol (U-CyTech, Utrecht, The Netherlands),Spots were counted on an Olympus microscope and analyzed with OlympusMicro Image 4.0 software (Paes Nederland, Zoeterwoude, The Netherlands).The final results are expressed as fraction of positive stimulationindices (positive: >2).

ERK-½ activation was tested with cells from the muco-epidermoid lungtumor cell line NCI-H292 (ATCC, Rockville, Md.), which were cultured in24- or 6-well tissue culture plates in RPMI1640 medium (Gibco, GrandIsland, N.Y.) supplemented with 2 mM L-glutamine (Bio Wittaker,Walkersville, Md.), 200 U/ml penicillin (Bio Wittaker), 200 μg/mlstreptomycin (Bio Wittaker) and 10% (v/v) heat-inactivated fetal calfserum (Gibco). After reaching near-confluence, cells were culturedovernight in serum-free medium. Cells were subsequently stimulated for15 minutes with indicated stimuli. Cellular lysates were prepared usinglysisbuffer (0.5% [v/v] Triton X-100, 0.1M Tris-HCl pH 7.4, 100 mM NaCl,1 mM MgCl₂, 1 mM Na₃VO₄, mini complete protease inhibitor cocktail[Boeringer Mannheim, Roche, Basel, Switzerland]). Samples were subjectedto SDS-PAGE on a 10% glycine-based gel, and resolved proteins weretransferred to a polyvinylidene difluoride (PVDF) membrane. Non-specificbinding sites were blocked by PBS/0.05% Tween-20/1% casein. The blotswere incubated with rabbit polyclonal antibodies against phosphorylatedERK-½ (New England Biolabs, Beverly, Mass.), and secondary horseradishperoxidase conjugated anti-rabbit IgG antibodies. The enhancedchemoluminescent (ECL) Western blotting detection system (AmershamPharmacia Biotech, Upsala, Sweden) was used to reveal immunoreactivity.

Neutrophils chemotaxis was measured with neutrophils isolated fromperipheral blood using Percoll density centrifugation (density: 1.082g/ml). The cells were resuspended at a concentration of 2.5×106 cells/mlin chemotaxis medium (20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer)HEPES, 132 mM NaCl, 6 mM KCl, 1.2 mM KH₂PO₄, 1 mM MgSO₄, 5.5 mM glucose,0.1 mM CaCl₂ and 0.5% (wt/vol) human serum albumm [Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam,The Netherlands] diluted 1:1 with serum-free RPMI. The chemotacticactivity of the compounds was assessed using a modified Boyden Cambertechnique. Briefly, 26 μl stimuli diluted in HEPES buffer was added tothe wells of the lower compartment, and 50 μl of neutrophil suspension(2.5×10⁶ cells/ml) was added to the upper compartment. The compartmentswere separated by two filters: a lower filter with a pore size of 0.45μm (Millipore Products, Bedford, Mass.) and an upper filter with a poresize of 8 μm (Sartorius Filter, San Francisco, Calif.). After incubationfor 90minutes at 37° C., the upper filters were removed, fixed inethanol-butanol (80:20, vol/vol), and stained with Weigert solution. Todetermine neutrophil chemotactic activity, neutrophils were counted insix random high-power fields (×400), and the percentage neutrophils onthe membrane as compared to the positive control (10⁻⁸ M,N-formylmethionyl-leucyl-phenylalanine (FMLP, Sigma) was calculated.

The results are given in table 2. In summary, the tested compounds ofthe invention, and in particular P60.4, induced a very low immuneresponse, lower than the natural peptide LL-37. They showed a lowERK-activation and virtually no neutrophil chemotaxis.

TABLE 2 Immunogenicity of compounds γ-IFN T cell ERK- Compound Elispotproliferation activation Chemotaxis (%) P60 1/8 7/8 − 76 ± 39 P60.Ac 3/84/8 ± 61 ± 36 P60.4 3/8 2/8 ± 0 ± 0 P60.4Ac nd nd ± 0 ± 0 LL-37(control) 4/8 6/8 + 84 ± 17

EXAMPLE 4 In-vivo Tolerability

Compound P60.4Ac was prepared according to example 1 and tested for itstolerability in vivo. More specifically, its potential for causing skinand eye irritation was evaluated in rabbits, whereas its ototoxicity wasstudied in a guinea pig model. Furthermore, its systemic toxicity wasassessed after intravenous administration.

For the skin and eye irritation tests, three rabbits were exposed to 0.5ml phosphate buffered peptide solution (2 mg/ml), applied onto clippedskin for 4 hours using a semi-occlusive dressing. Observations were made1, 24, 48, and 72 hours after exposure. Single samples of 0.1 ml ofphosphate buffered (pH7.5) peptide solution (2 mg/ml) were instilledinto one eye of each of three rabbits to perform an acute eyeirritation/corrosion study. Observations were made 1, 24, 48, and 72hours after instillation.

In result, no skin irritation was detectable. Ophthalmic instillation ofthe peptide solution resulted in redness of the conjunctivae whichresolved completely within 24 hours after instillation.

The systemic toxicity of P60.4Ac was assessed in a single and repeateddose toxicity study in rats. The peptide was administered dailyintravenously in escalating doses. In this phase, the Maximum ToleratedDose (MTD) was established. Repeated dose toxicity was also studied inthe MTD phase. In the dose escalation phase, 9 rate were divided inthree groups and received 0.4, 2 or 8 mg/kg/day for two days. Clinicalsigns were recorded twice daily on days of dosing and one day afterdosing, body weights were recorded prior to the first dose and one dayafter dosing. In the MTD phase, 5 female and 5 male rats received 8mg/kg/day for 5 following days. Clinical signs were recorded twice dailyon days of dosing, body weight on day 1 and 6. Clinical laboratoryinvestigations were performed prior to necropsy. Macroscopy wasperformed at termination of the MTD phase.

In result, no mortality occurred in the systemic dose escalation study.Furthermore, no clear deviations were noted in clinical signs and bodyweight. During the MTD phase also no mortality occurred and no clearpeptide related findings were noted in clinical signs, body weight,hematology and clinical biochemistry parameters and at macroscopicexamination.

EXAMPLE 5 Ototoxicity

To evaluate the ototoxicity of peptide P60.4-Ac, this peptide was testedin guinea pigs (HsdPoc.DH; Harlan, Horst, The Netherlands). Sevenhealthy male albino guinea pigs (500-1200 g), free of external earpathology, were used in this study. Animals were anesthetized withintraperitoneal injections of 40 mg/kg ketamine (Eurovet Animal HealthB.V., Bladel, The Netherlands) and 10 mg/kg rompun (Bayer A.G.,Leverkusen, Germany). After control auditory testing was performed, theauditory bullae were surgically opened to apply a small piece ofspongostan to the round window membrane (RWM) and various solutions(approximately 10 μl) were added on the spongostan. The skin was suturedclosed and follow-up auditory testing was performed. Application on theRWM was performed in the right ears, the left ears remained untreated.One animal received PBS as a first placebo solution (placebo I), andanother animal received the second placebo solution (placebo II), whichwas 7% Macrogol 10 000 in isotonic [NaCl] and preserved [0.02%benzalkonium chloride and 0.1% Na₂EDTA] 20 mM phosphate buffer solution(pH 5.5). Two guinea pigs received cisplatin (0.66 mg/ml in PBS,obtained from Sigma Chemicals, Zwijndrecht, The Netherlands), whichserved as a positive control for the test [39]. Peptide P60.4-Ac (2mg/ml) was tested in PBS solution (formulation I) in one animal and in abuffer corresponding to the second placebo (formulation II) in two otheranimals.

Auditory brainstem response (ABR) was performed prior to drugadministration and directly after surgery and 3, 7, 14 and 22 dayslater, using a computer-based signal averaging system (Tucker-DavisTechnology, Alucha, Fla., USA). Guinea pigs were anesthetized and aninsert earphone was placed into the external ear canal. Subcutaneouselectrodes were placed over the vertex (active) and over the ipsilateralbulla (reference). Ground electrodes were placed over the neck muscles.ABRs were recorded in an electrically shielded, double-walled,radio-frequency-shielded sound chamber in response to 10 ms tone burstsat 1 kHz. Stimulus intensities were measured and expressed as dB. ABRthreshold was defined as the lowest intensity capable of eliciting areplicable, visually detectable response. The post-treatment ABRthresholds were compared to pre-treatment ABR thresholds.

In result, round window application of PBS, which was used as a control,did not result in a threshold change at 22 days after surgery.Formulation buffer resulted in a threshold change of 2 dB after 22 days.Cisplatin, on the other hand, induced threshold changes of respectively−49 dB and −64 dB, which indicate a severe hearing loss (Table 3). Thispart of the experiments served as a positive control for the ototoxicitystudy. Peptide P60.4-Ac (2 mg/ml) in PBS induced a threshold change of−7 dB 22 days after surgery. Both animals that received P60.4-Ac asformulation II produced a threshold change of 1 dB.

TABLE 3 Results of ototoxicity evaluation of P60.4Ac P60.4-Ac PlaceboP60.4-Ac Placebo Formulation Group 1 I^(a) Cisplatin Formulation I^(a)II^(b) II^(b) post- 1 −2 −1 −2   1 14 20 surgery  3 days −3 −32 −38 −2−8 −1 1  7 days −3 −32 −45 0 −33^(c)   −18 2 14 days 0 −30 −59 −12 −1 02 22 days 0 −49 −64 −7   2 1 1 Values represent Δ presurgery in dB.^(a)PBS ^(b)7% Macrogol 10.000 in isotonic [NaCl] and preserved [0.02%benzalkonium chloride and 0.1% Na₂EDTA] 20 mM phosphate buffer solution(pH 5.5) ^(c)Unreliable measurements due to bad wire

EXAMPLE 6 Antimicrobial Activity

Compound P60.4Ac was prepared according to example 1 and sterilised bysterile filtration into 10 mL glass bottles with screw closures. Nopreservative was added. Separately, a corresponding placebo solution,i.e, a solution with the same components except for compound P60.4Ac,was prepared. Samples were drawn from each of the two preparations forconducting a test for antimicrobial activity. In result, the solutioncontaining compound P60.4Ac was found to inhibit bacterial growth oreven reduce the number of germs, whereas the corresponding placebosolution showed no antimicrobial activity.

EXAMPLE 7 Antimicrobial Activity

The in vitro antibacterial and antifungal activity of P60.4-Ac and LL-37were determined as the minimum inhibitory concentrations (MIC) by amicrodilution susceptibility test in 96-well microtiter plates,according to a modified version of R. Hancock's “Modified MIC method forcationic antimicrobial peptides” [13]. The antibacterial activity wastested on the reference strains Escherichia coli ATCC 8739, Pseudmonasaeruginosa ATCC 9027. The antifungal activity was evaluated on Candidaalbicans ATCC 10231 and Aspergillus niger ATCC 14406. The antimicrobialactivity assay was conducted with different concentrations of P60.4Acand LL-37 to compare their effects on the bacterial or fungal growth.Antibacterial activity was examined using log-phase cultured bacteria inTrypticase Soy Broth at 37° C. The cultures were diluted with 10 mMSodium Phosphate buffer pH 7.4 to give approximately 5.0×10⁶ CFU/ml. 10μl of the diluted test strain was transferred to a 96-well plate and 100μl of the different peptide concentrations was added to each well. Theplates were incubated at 37° C. for 24 hours and are then scored forgrowth by visual inspection on a light box. They are then returned forincubation for a further 24 hours after which time they are re-evaluatedfor growth. The yeast strain C. albicans was prepared as describedabove. The filamentous fungi A. niger was used as a spore suspension,cultured on Sabourauds Dextrose Agar plates at 20-25° C. for 6-10 daysor until adequate sporulation has occurred. The spores were harvested byscrapping and if necessary the concentration was adjusted to a finalconcentration of 5×10⁶ CFU/ml.

Thus, the antimicrobial activity of P60.4-Ac was evaluated against twoGram-negative strains and against the fungi C. albicans and A. niger andcompared with LL-37. The MIC values for each peptide are given in Table4. P60.4-Ac showed a higher or equal activity against the Gram-negativestrains E. coli and P. aeruginosa compared to LL-37. In some cases alsobactericidal activity was determined for both peptides. P60.4-Ac showedan MIC at 6 μM against C. albicans and may well be fungicidal at 18 μM.P60.4-Ac at 18 μM inhibited the germination of A. niger spores for 24hours, whereas LL-37 shows no activity against A. niger.

EXAMPLE 8 Inhibition of Biofilm Formation

Compound P60.4Ac was prepared according to example 1 and tested for itseffectiveness as inhibitor of Pseudomonas putida PCL1445 biofilmformation. A standard PVC biofilm assay was used, in which biofilms areformed on the polyvinyl chloride surface of the wells of microtiterplates. To the P. putida suspension in the wells, a solution of P60.4Ac(0.9 μM and 9 μM) was added and incubatedfor 10 hours. In result, it wasfound that P60.4Ac at 9 μM inhibited biofilm formation by more than 90%,whereas 0.9 μM resulted in a decrease of approx. 50% compared withbuffer solution without the peptide.

EXAMPLE 9 Inhibition of Formed Biofilms

Compound P60.4Ac was prepared according to example 1 and tested for itseffectiveness in disrupting Pseudomonas putida biofilms. The biofilmswere formed using a standard PVC biofilm assay as in example 7. Biofilmswere allowed to form in the wells of the microtiter plate from P. putidaPCL1445 suspensions over 7 hours. After that, different quantities ofP60.4Ac were added, as well as controles, i.e. DMSO and the bufferedmedium solution, M63, respectively. After 18 hours of incubation, thebiofilms were assessed via their optical density at 595 nm (OD595). Inresult, DMSO resulted only in a small reduction of OD595 in comparisonwith M63, whereas P60.4Ac disrupted the biofilms substantially,depending on its concentration.

FIG. 1 shows the OD595 values for the various molar concentrations ofP60.4Ac (bars 1 to 5), DMSO and M63,

TABLE 4 Antimicrobial activity of P60.4-Ac compared with LL-37 Organism(strain) Incubation time (h) Peptide MIC^(a) (μM) E. coli 24 LL-37  3ATCC8739 P60.4-Ac  2 48 LL-37  7^(b) P60.4-Ac  3^(c) P. aeruginosa 24LL-37  3 ATCC9027 P60.4-Ac  3 48 LL-37  14^(d) P60.4-Ac  6^(b) C.albicans 24 LL-37  12 ATCC10231 P60.4-Ac  6 48 LL-37 >18^(e) P60.4-Ac 6^(f) A. niger 24 LL-37 >18 ATCC14406 P60.4-Ac  18 48 LL-37 >18^(e)P60.4-Ac >18^(e) ^(a)MIC was defined as the lowest concentration ofpeptide that inhibited the bacterial visible growth after incubation for24 or 48 hours at 37° C. Results given are mean values of threeindependent determinations. ^(b)Bactericidal at 18 μM ^(c)Bactericidalat 6 μM ^(d)Bacteriostatic ^(e)Recovery of viable organisms was notperformed, as growth in all wells was clearly visible ^(f)Possiblefungicidal at 18 μM, fungistatic at 6 μM

1. A method to inhibit the formation of, or disrupt or degrade biofilmsin a mammal, which method comprises administering to a mammal in need ofsuch treatment an effective amount of a compound of formula (1) thatcomprises a core amino acid sequence: X₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆ (SEQ IDNO;1), wherein X₁ represents an N-terminal part; X₂ is K or E; X₃ is Qor E; X₄ is D or R; X₅ is N or E; X₆ represents a C-terminal part;wherein one or more of the amino acids of the core amino acid sequenceis optionally derivatized, and wherein (a) the N-terminal part isacetylated, and/or (b) the C-terminal part is amidated, and/or (c) thecore amino acid sequence is different from KEFKRIVQRIKDFLRNLV.(SEQ ID NO: 2)


2. The method according to claim 1, wherein said biofilms are caused bysessile, biofilm-forming microorganisms.
 3. The method according toclaim 1, wherein the biofilms are associated with periodontitis, nativevalve endocarditis, cystic fibrosis, chronic bacterial prostatitis,bronchitis, pneumonia, sinusitis, dental caries, chronic tonsillitis,endocarditis, necrotizing fasciitis, musculoskeletal infection,osteomyelitis, biliary tract infection, or infectious kidney stones. 4.The method according to claim 1, wherein the biofilms are associatedwith an infection of the liver, the spleen, the periodontium, an eye, akidney, the skin, the vagina, the urethra, or the heart.
 5. The methodaccording to claim 1, wherein the compound is present in a fluid at aconcentration of at least 0.001 μM.
 6. The method according to claim 5,wherein the compound is present in the fluid at a concentration in therange from 0.1 to 100 μM.
 7. The method according to claim 1, whereinthe compound is formulated and processed for parenteral injection,instillation or irrigation.
 8. The method according to claim 1, whereinthe compound is formulated substantially free of preservatives.
 9. Themethod according to claim 1, wherein the N-terminal part X₁ comprisesthe amino acids I and/or G.
 10. The method according to claim 1, whereinthe C-terminal part X₆ comprises a sequence of at least 4 amino acids oramino acid derivatives.
 11. The method of claim 1, wherein theC-terminal part X₆ comprises PRTE or RPLR, wherein one or more of theamino acids of said C-terminal part X₆ is optionally derivatized. 12.The method of claim 1, wherein the compound comprises a peptide with asequence of 24 amino acids or derivatives thereof, said sequence beingselected from IGKEFKRIVQRIKDFLRNLVPRTE (SEQ ID NO:3) andIGKEFKRIVERIKRFLRELVRPLR (SEQ ID NO:4), and wherein one or more of theamino acids is optionally derivatized.
 13. The method of claim 12,wherein the N-terminus is acetylated and the C-terminus is amidated. 14.A method to prevent or manage an infection resulting from insertion orimplantation of a medical device in the body of a mammal for adiagnostic or therapeutic purpose which method comprises administeringto a mammal in need of such treatment an effective amount of a compoundof formula (1) that comprises a core amino acid sequence:X₁KEFX₂RIVX₃RIKX₄FLRX₅LVX₆(SEQ ID NO:1), wherein X₁ represents anN-terminal part; X₂ is K or E; X₃ is Q or E; X₄ is D or R; X₅ is N or E;X₆ represents a C-terminal part; wherein one or more of the amino acidsof the core amino acid sequence is optionally derivatized, and wherein(a) the N-terminal part is acetylated, and/or (b) the C-terminal part isamidated, and/or (c) the core amino acid sequence is different fromKEFKRIVQRIKDFLRNLV. (SEQ ID NO: 2)


15. The method according to claim 14, wherein the medical device is acontact lens, a heart valve, a venous catheter, a urinary catheter, aspeech button, a tympanostomy tube, an intrauterine device, a suture, avascular graft, a vascular shunt, a peritoneal dialysis deice, a penileprosthesis, an orthopaedic prosthesis, or an artificial bone.
 16. Themethod according to claim 14, wherein the compound is present in a fluidat a concentration of at least 0.001 μM.
 17. The method according toclaim 16, wherein the compound is present in the fluid at aconcentration in the range from 0.1 to 100 μM.
 18. The method accordingto claim 14, which compound is formulated and processed for parenteralinjection, instillation or irrigation.
 19. The method according to claim14, wherein the compound is formulated substantially free ofpreservatives.
 20. The method according to claim 14, wherein theN-terminal part X₁ comprises the amino acids I and/or G.
 21. The methodaccording to claim 14, wherein the C-terminal part X₆ comprises asequence of at least 4 amino acids or amino acid derivatives.
 22. Themethod of claim 14, wherein the C-terminal part X₆ comprises PRTE orRPLR, wherein one or more of the amino acids of said C-terminal part X₆is optionally derivatized.
 23. The method of claim 1, wherein thecompound comprises a peptide with a sequence of 24 amino acids orderivatives thereof, said sequence being selected fromIGKEFKRIVQRIKDFLRNLVPRTE (SEQ ID NO:3) and IGKEFKRIVERIKRFLRELVRPLR (SEQID NO:4), and wherein one or more of the amino acids is optionallyderivatized.
 24. The method of claim 23, wherein the N-terminus isacetylated and the C-terminus is amidated.